International Journal of

Molecular Sciences

Review

Legume Lectins: Proteins with Diverse Applications

Irlanda Lagarda-Diaz 1, Ana Maria Guzman-Partida 2 and Luz Vazquez-Moreno 2,*1 CONACyT-Universidad de Sonora, Blvd. Luis Encinas y Rosales, Hermosillo, Sonora 83000, Mexico;

Irlanda.lagarda@unison.mx2 Coordinacion de Ciencia de los Alimentos, Centro de Investigacion en Alimentacion y Desarrollo, A.C.,

Apartado Postal 1735, Hermosillo, Sonora 83304, Mexico; gupa@ciad.mx* Correspondence: lvazquez@ciad.mx; Tel./Fax: +52-662-280-0058

Academic Editor: Els Van DammeReceived: 29 April 2017; Accepted: 5 June 2017; Published: 12 June 2017

Abstract: Lectins are a diverse class of proteins distributed extensively in nature. Among theseproteins; legume lectins display a variety of interesting features including antimicrobial; insecticidaland antitumor activities. Because lectins recognize and bind to specific glycoconjugates presenton the surface of cells and intracellular structures; they can serve as potential target molecules fordeveloping practical applications in the fields of food; agriculture; health and pharmaceutical research.This review presents the current knowledge of the main structural characteristics of legume lectinsand the relationship of structure to the exhibited specificities; provides an overview of their particularantimicrobial; insecticidal and antitumor biological activities and describes possible applicationsbased on the pattern of recognized glyco-targets.

Keywords: lectin; legume; insecticidal; antimicrobial; cancer

1. Introduction

Currently, lectins are defined as carbohydrate binding proteins of non-immune origin that canrecognize and bind simple or complex carbohydrates in a reversible and highly specific manner.For a long time, these proteins were named hemagglutinins, due to their ability to agglutinate redblood cells. At present, this concept is used when the specificity is unknown. It is also known thatmonomeric lectins do not exhibit this agglutinating activity [1]. Lectins are ubiquitously presentin fungi, animals, bacteria, viruses and plants. Among plant lectins, those of legumes have beenthe most widely considered [2]. These lectins are abundant in the seeds and belong to a group ofhighly homologous proteins. However, their carbohydrate specificities and quaternary structures varyextensively [3], which greatly influences the type of recognized targets.

Throughout the history of lectin research, legume seeds have been screened for lectinactivity. The existence of binding sites for specific carbohydrates, the main characteristic of lectins,is undoubtedly an important factor for determining their activity and future applications based on theirproperties. This review centers on the lectins of legumes, highlighting their specificity, particularities,main reported activities and potential applications.

2. Structure of Legume Lectins

Legume lectins represent the largest family of carbohydrate binding proteins, and theirphysicochemical and biological properties have been broadly studied [4–8]. Research of the plantlectin family using newer biochemical and biophysical strategies has significantly moved the fieldforward, and provided a model framework for studying protein–carbohydrate interactions. Althoughplant lectins share high sequence homology and exhibit comparable physicochemical and structuralproperties, the diversity of their carbohydrate recognition specificity is remarkable. Generally,

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the three-dimensional structure of plant lectins is characterized by β-sheets that are connected byα turns, β turns and bends. In addition to short loops, the quaternary interfaces are formed betweenβ-sheets. The β-sheets are connected by loops forming antiparallel chains usually devoid of α helices(Figure 1) [4,5,9–12].

Figure 1. Overall structure of legume lectins. (A) tetramer with carbohydrate recognition domains(red); (B) amplified image of monomer with β-sheets (yellow), α-turns (purple), metal binding sites(area with green and gray spheres) and carbohydrate recognition domain (area occupied by the greyand red molecule). Image modified from Protein Data Bank (accession code 1GZ9).

Analysis of legume lectin monomers reveals high similarity in sequence as well as structure, withonly minor variations in the length of loops and strands. The monomer structures display a jellyrollmotif best described as a sandwich of 25–30 kDa containing a carbohydrate recognition domain (CRD)and metal binding sites for divalent cations (Ca2+ and Mn2+). However, in spite of strong similaritiesin primary, secondary and tertiary structures, the quaternary structures show considerable variationsthat lead to changes in the monomer–monomer interactions and the presence/absence of proteinmodifications, such as glycosylation. The impact of these variations has functional implications asthey dictate the specificity of multivalent glycan binding [1,5,11,13–18]. Most legume lectins appear toassemble as homodimers or homo-tetramers (dimers of dimers), the stability of which is attributed tohydrophobic cooperation, hydrogen bonds and salt links [4,9,19].

Legume lectins are subdivided into two categories, those with indistinguishable or almostindistinguishable subunits, and those with a variety of subunits [20]. Phaseolus vulgaris lectins (PHA)are well-studied lectins of the first group [21–26]. There are more than forty structures of legume lectinsreported in either the apo (ligand-free) or holo (sugar-bound) form in the Protein Data Bank [15,26–32].However, data about lectins from wild legumes is rare.

Mexico has roughly 1800 species of wild legumes confined to various regions, and more than33 percent are within the Sonoran Desert [5,33–42]. The isolation and purification of lectins fromseeds of native plants such as Parkinsonia aculeata, Olneya tesota, Acacia constricta, Prosopis juliflora,Cercidium praecox, Caesalpinia caladenia and Phaseolus acutifolius has been described. Lectins from theseplants have monomers of either 30 or 66 kDa that also can form tetramers or dimers. In addition, it hasbeen shown that these lectins involve a group of different isolectins, compared to those found in lectinsof the Phaseolus genus [43–47].

On the other hand, the primary sequences of plant lectins have been used to infer evolutionaryrelationships, to reflect processing routes, to suggest folding patterns as well as to provide clues on howcarbohydrate ligands fit into their binding pockets [20]. Direct amino acid sequencing has revealed

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homology among the lectins from the wild desert legumes A. constricta [6] and O. tesota [5,48], and moredomesticated species like P. vulgaris and P. coccineus [49,50]. Furthermore, antibodies raised againstPHA show reactivity with lectins from A. constricta, C. praecox and C. caladenia [6]. From these studies,it is clear that lectins have been conserved during the evolution of legumes and that the extensivehomologies reflect taxonomical relationships among these plants [51].

3. Specificity of Legume Lectins

Legume lectins represent a crucial point in the study of the molecular basis of protein–carbohydrateinteractions. Each has a CRD with a basic architecture accessible to both monosaccharidesand/or oligosaccharides that confers remarkable divergence in their specificities [12,52]. Lectins cannon-covalently interact with carbohydrates in a manner that is usually reversible and highly specific.Recognition can occur in the terminal or intermediate position of the glycan structure [53]. In all legumelectins, four invariant amino acid residues participate in the binding of the carbohydrate, yet eachlectin shows different specificity [18,54,55]. The CRD pocket is formed by four loops adjacent to eachother, but the loops are not close in sequence. These regions exhibit the highest residue variabilityand appear to be involved in specificity determination [56,57]. The CRD of legume lectins lies in closeproximity to the metal binding sites, which may aid in binding activity [18], but are not often directlyinvolved in carbohydrate binding [18].

Most lectins bind to mono- and oligosaccharides; however, more recently discovered lectins showspecificity for complex sugars and glycoproteins. Generally, lectins show specificity for di-, tri- andtetra-saccharides with association constants significantly higher than those for the correspondingmonosaccharides [5,18,58]. Some lectins have shown affinity towards carbohydrate structures notpresent in plants, such as the Thomsen-nouveau antigen or complex N-glycan structures with terminalgalactose and sialic acid residues [30]. The specificity of legume lectins for some typical animal glycans,has led to the suggestion that legume lectins play a role in plant defense against insects and/or predatoranimals [59,60].

The legume lectins that have been purified and characterized as members of the complex typeinclude, Griffonia simplicifolia IV lectin (GS IV) [5,61], Maackia amurensis lectin (MAH and MAL-1) [62],Cicer arietinum lectin [63], Saraca indica lectin [58,64] and P. vulgaris E and L lectins (PHA-E andPHA-L) [5,23,26,65–69]. Wild legume lectins from the Sonoran Desert also have shown varyingdegrees of specificity for some complex carbohydrates of glycoproteins. The hemagglutinatingactivity of A. constricta isolectins (VLs) and the P. juliflora lectin is strongly inhibited in the presenceof asialofetuin and to a lesser extent with fetuin and thyroglobulin [6,40]. C. praecox and C. caladenialectins are inhibited by fetuin and mucine [37]. O. tesota PF1 and PF3 lectins are inhibited by freemonosaccharides and/or by complex carbohydrates of fetuin, thyroglobulin, immunoglobulin, mucineand ovalbumin, whereas PF2 is only inhibited by the intact glycoprotein [5]. Although glycan structurespresent in glycoproteins are known to have N and O linked oligosaccharides, desialylation of the glycancan result in an increase or decrease in lectin binding affinity [70]. The galactose residues foundin glycosylated proteins are often capped with sialic acid, a characteristic that may alter the interactionwith lectins, as is seen for Amaranthus caudatus and Abrus pulchellus [71,72].

Many studies on lectin–carbohydrate specificity have been published. Unfortunately, oftenspecificity analyses are used to “specifically” determine structural motifs in glycoconjugates withouttaking into account the rather complex data on this subject. Furthermore, the choice of ligandsused to assay lectin binding in some studies is limited either by natural or commercially availablesources of glycans, or to simple mono- or disaccharides. Therefore, a critical re-evaluation oflectin binding specificities is required. Such studies using microarray techniques with immobilizedglycans or immobilized lectins, dot blot assays to screen different lectins and antibodies withmultiple oligosaccharides, combined with information gleaned using recombinant glycosyltransferases,are opening up new possibilities of generating a broader range of standard (neo)glycoconjugates withclearly defined structures [73].

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4. Antimicrobial Activity

Antimicrobials have been used to treat diseases caused by bacteria and fungi and such treatmentshave significantly reduced the mortality rate from these infections in humans and animals. However,the extensive use of antimicrobial substances in medical, agricultural, and veterinary practices is a topicof great concern to clinical microbiologists all over the world due to the growing emergence ofopportunistic microorganism strains resistant to drugs that cause serious infections [74–77]. Microbialresistance is a genetic phenomenon resulting in a reduction in effectiveness of drug therapy; it maybe caused by mutations during the reproductive process of the microorganisms or by importedgenes acquired through transduction, conjugation, and transformation mechanisms [78]. Therefore,there is a growing interest in developing new strategies for inhibiting the growth or survival ofmicroorganisms based on the screening of new sources of natural compounds as alternatives tocommercially available drugs [74,79].

A large number of proteins with antibacterial, antifungal and/or antiviral activity have beenisolated from the seeds, tubers, and rhizomes of different plants, where they accumulate to highlevels and may function as a reserve source of amino acids and metal ions [80]. These proteinsmay directly interfere with the growth, multiplication and spread of microbial agents by differentmechanisms [81], as in the case of lectins, by agglutination and/or microorganism immobilization.Plant lectins often are present at potential sites of microbial invasion and their binding to fungalstructures led to the inhibition of fungal growth and germination. Studies carried out with soy beanand common lentil agglutinins provide evidence for these roles [51,82–84]. A main characteristicof lectins is their ability to interact with glyco-components present on the cell membrane surface,in cytoplasmic and nuclear structures and in the extracellular matrix of cells and tissues from almost allkingdoms of life [85]. Therefore, plant lectins are thought to play important roles in the plant immunedefense and emerge as potential antimicrobial candidates for drug therapies. The antimicrobial effectof legume lectins is shown in Table 1. Although many lectins show antimicrobial activity, clinical trialsare needed to establish therapeutic efficacy, to optimize dosage, delivery and bioavailability, and toassess potential allergic reactions [86].

4.1. Bacteria

The antibacterial activity of lectins results from their interaction with a wide variety of complexcarbohydrates of the bacterial cell wall, such as teichoic and teichuronic acids, peptidoglycans andlipopolysaccharides [2,85]. Lectins of Triticum vulgare, Dolichos lablab L., Trigonella foenumgraecum,Trifolium alexandrium L., Bauhinia variegata L. and Delonix regia have shown antimicrobial activityagainst Mycobacterium rhodochrous, Bacillus cereus, B. megaterium, B. sphaericus, Escherichia coli, Serratiamarcescens, Corynebacterium xerosis and Staphylococcus aureus [86] Lectins from the Vicieae tribe stronglyreact with the bacterial cell wall components, muramic acid, N-acetylmuramic acid and muramyldipeptides [87]. Nevertheless, although legume seed lectins can recognize and bind to the constituentsof the bacterial cell wall, such binding does not imply that these interactions occur in vivo, and certainlydoes not prove that these lectins are involved in the protection of seedlings against bacteria [51].

It is thought that plant lectins do not alter the membrane structure and/or permeability nordisturb the normal intracellular processes of invading microbes, since they exert an indirect effect thatis based on interactions with cell wall carbohydrates or extracellular glycans [60]. However, electronmicroscopy studies revealed that treatment with Araucaria angustifolia lectin promoted morphologicalterations, including the presence of pores in the membrane of Gram-positive bacteria, and bubblingon the cell wall of Gram-negative bacteria [88]. Others reported that the antibacterial activity of lectinsoccurred through the interactions of the lectins with N-acetylglucosamine, N-acetylmuramic acid andtetrapeptides linked to N-acetylmuramic acid present in the cell wall of Gram-positive bacteria, or tolipopolysaccharides present in the cell wall of Gram-negative bacteria [87].

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Table 1. Antimicrobial effect of legume lectins.

Lectin Carbohydrate/Glycoprotein Receptor Type ofMicroorganism Mechanism of Action Reference

From Leguminosae,tribe: Vicieae

DiocleaePhaseoleaeErithrineaGlycineaeSophoreaeGalegeaeGenisteae

LoteaeAcacieae

Components of the cell Wall: Muramic acid,N-acetylmuramic acid,

N-acetylglucosamine, Muramyl-dipeptides,glucosaminyl-muyamyl-dipeptide,

lipopolysacharides

Bacteria

Form a channel or pore on cellmembrane and the cell dies as a result of

the out flowing of cellular contents.Bacterial aggregation and inhibition

bacterial cell division

[79,83,87,89–91]

Astragalusmongholicusagglutinin

P. vulgaris agglutininP. coccineusagglutinin

Soy bean agglutininPeanut agglutinin

Components of fungal cell wall: Chitin,sialic acid Fungi

Binding to hyphae, swollen hyphal,vacuolization of the cell content, and

enhanced susceptibility to cell wall lysisof the hyphal induced by osmotic shock,producing more susceptibility to other

stress conditions. This conditionproduces poor absorption of nutrients,

interference spore germination andrupture of the cell wall.

[79,83,91–98]

Concanavalin APsophocarpustetragonolubus

agglutininLens culinaris

agglutininVicia faba agglutinin

Pisum sativumagglutinin

Erythroagglutinin

Components of viral envelope:Glycoproteins Gp120/Gp41, sialic acid Virus

Bind to the glycosylated envelopeprotein and block cellular entry (interfere

with replication cycle)[98–105]

In addition to these findings, lectins from the legumes from Canavalia ensiformis, T. foenumgraecum,Arachis hypogaea, Cajanus cajan, P. vulgaris and Pisum sativum were shown to inhibit biofilm formationby Streptococcus mutans, but the growth of planktonic cells was not affected [89]. Since bacterialbiofilms make disinfection procedures more difficult by increasing bacterial resistance to detergentsand antibiotics, the inhibition of biofilm formation and development by legume lectins could be a usefulcharacteristic of these proteins. Furthermore, the rapid evolution of bacteria with antibiotic resistance(in particular, multidrug resistant bacteria or superbugs) has reduced the efficacy of conventionaltreatments against their biofilms. Hence, development of non-antibiotic alternatives that couldefficiently treat or reduce biofilms should become a priority. Furthermore, research focused on findinglectins that reduce superbugs could represent an interesting alternative drug therapy that deservesfurther attention.

4.2. Fungi and Yeasts

Regardless of the considerable number of lectins that have been characterized, only a small numberhave shown antifungal effects. Because plant lectins do not penetrate the cell wall or the membraneto reach the cytoplasm of fungi, direct inhibition of fungal growth by lectins is unlikely. In spite ofthis, indirect responses produced by the attachment of lectins to chitin and other glycans on the fungalsurface could affect fungal survival or other activities [105,106].

Lectin binding to hyphae could result in inhibition of fungi growth as a result of poor nutrientabsorption as well as by interference with the spore germination process [107]. Lectins can causedifferent morphological changes that render fungi more vulnerable to differing stress conditions [108].For example, in one study, lectin interactions resulted in swollen hyphae, vacuolization of the cellcontent and improved lysis of hyphal cell wall, that, in turn, increased susceptibility of fungi to osmoticshock [107]. In another case, it was suggested that small antifungal lectins could penetrate the fungalcell wall and reach the cell membrane where blocking of active sites of enzymes could alter cell wallmorphogenesis [108,109].

Some reports indicate that lectins from the legumes Astragalus mongholicus, P. coccineus, Archidendronjiringa Nielsen, B. ungulata, Glycine max, Indigofera heterantha and A. hypogaea can exhibit antifungalactivity against various species of phytopathogenic fungi, such as Botrytis cinerea, Fusarium oxysporum,

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F. moniliforme, F. solani, Colletorichum sp., Drechslera turcia, and Exserohilum turcicum, as well aspathogenic fungi such as Candida albicans, Penicillium italicummm, and Aspergillus sp. [82,91–93,110,111].Additional studies are required to elucidate the molecular basis for the antifungal activity of individuallectins as the ability of lectins to inhibit growth differs among fungal species.

4.3. Virus

Plant lectins with antiviral activity are of substantial therapeutic interest. Some of thesecarbohydrate binding proteins exhibit significant activity against human immunodeficiency virus(HIV) and other viruses [97]. Retroviruses, such as HIV, have a surface covered by highly glycosylatedvirally-encoded glycoproteins, e.g., gp120 (that contains high mannose and/or hybrid glycans) andgp41. The carbohydrates on these proteins are particularly important because the glycans canbe used as tools to render the virus easily recognized by the immune system, and thus susceptible toimmunological neutralization [112]. The interactions between host and proteins from the viral envelopalso can be altered by compounds that specifically recognize and bind glycans. Furthermore, lectinscan crosslink surface viral glycans and thereby prevent interactions with other co-receptors [83].

Antiviral lectins used as therapeutic agents offer lower toxicity and can be included in topicalapplications. Generally, lectins are odorless, and resistant to high temperatures and low pH, idealproperties for development of microbicide drugs [86].

Most current antiviral therapeutics prevent the viral life cycle, or inhibit the entrance ofthe virus into host cells [113]. The antiviral action of plant lectins varies extensively dependingupon their carbohydrate specificity. Corona virus are highly susceptible to mannose specific lectinsthat interfere with the viral attachment in early phases of the replication cycle and suppress viraldevelopment by binding towards the end of the viral infection cycle [114]. Other antiviral non-legumelectins with encouraging properties include griffithsin (GRFT), cyanovirin (CV-N) and banana lectin(BanLec), and BanLec has been recommended as an antiviral microbicide. Most often antiviral lectinsare recommended for incorporation into vaginal and rectal gels, creams or suppositories that act asa barrier to prevent HIV transmission. In such processes, lectins bind the virus preventing its entryand fusion to target cells, consequently averting contamination [115].

Several legume lectins possess antiviral activity. Lectins like C. ensiformis agglutinin (Con A),Lens culinaris agglutinin (LCA), Vicia faba agglutinin, P. sativum agglutinin (PSA) and PHA-E bindto the envelope glycoprotein gp120 and to inhibit fusion of HIV-infected cells with CD4 cells bya carbohydrate-specific interaction with the HIV-infected cells [103]. In addition, G. max agglutininsinhibit HIV-1 reverse transcriptase activity [116]. Although glycan structures of viral proteins involvehigh mannose, further research in this area is needed to explore lectins that recognize structures withdifferent sugars such as sialic acid, fucose and N-acetyl glucosamine with the aim of developing novelapproaches and therapies in the field of virus biology.

5. Insecticidal Activity

Legume lectins are toxic to a broad spectrum of insects representing several orders including,Coleoptera, Diptera, Lepidoptera, Hymenoptera, Isoptera, Neuroptera and Homoptera. In this regard,the family of leguminous lectins is the most studied due to its insecticidal potential. A great number oflegume lectins have shown insecticidal activity including those from C. ensiformis, P. sativum, P. vulgaris,Glechoma hederacea, G. simplicifolia, O. tesota (PF2) and B. monandra [8,117,118].

These lectins showed harmful effects in the different developmental stages of insects (larvae andadults). In this regard, they may increase mortality, delay development and/or adult emergence andreduce fecundity. Usually, the effect of lectins on insects is evaluated by feeding larvae with artificialseeds containing the lectin or with transgenic plants that overexpress the lectin of interest. The expressionof a given lectin in transgenic plants is only possible if the lectin molecular sequence is well characterizedand also requires an available transformation protocol for the desired plant species [119].

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In the last twenty years, research has focused on elucidating the mode of action of insecticidallectins. In general, lectins can exert a toxic effect via binding to the peritrophic membrane (PM),peritrophic gel (PG) or the brush-border microvilli of epithelial cells (Figure 2) [120]. The PM orPG is a film surrounding the food bolus in most insects and is composed of chitin and proteins.Some non-legume lectins with insecticidal activity such as the Rhizoctonia solani agglutinin andSambucus nigra agglutinin II can pass through the PM of the red flour beetle, Tribolium castaneum.This ability to reach the endoperitrophic space is governed by the dimensions of the molecule andthe charge and size of the PM pores [121].

Figure 2. Insecticidal action mechanism for lectins.

In the case of insects lacking a PM, the insecticidal effect of lectins may be exerted by their directinteraction with glycoconjugates present on the cell epithelium [122]. In fact, the insecticidal activity oflectins relies on their binding properties to sugars present on the surface of the insect gut epithelial cells.In insects, the surface of epithelial cells is rich in glycoproteins that, in addition to their determinantroles for the proper gut function, provide a number of available targets for lectin binding.

The interaction of lectins with different glycoproteins or glycan structures in insects may interferewith important physiological processes (Table 2). Although the key receptors for lectins couldbe localized on the surface of gut epithelial cells, if lectins are internalized, they may interact with a newset of targets located in the intracellular space, allowing the lectin to interfere with particular metabolicpathways. Such internalization has been more studied for non-legume lectins. For example, the garlicleaf lectin, which affects survival and development of the moth Helicoverpa armigera, was found tobe internalized by binding to a glycosylated alkaline phosphatase anchored to the insect midgutmembrane. In addition, Galantus nivalis lectin was reported to cross the midgut epithelium ofNilaparvata lugens and co-localize in the fat body and hemolymph. Such co-localization was proposedto occur via a carrier-receptor, ferritin. The internalization of the Colocasia esculenta tuber agglutinin intoinsect hemolymph also was demonstrated, suggesting it could interact with various N-glycosylatedintracellular targets, which may, in part, explain the lectin toxicity [122]. Although the completemechanism by which some lectins are able to cross midgut epithelial cells is not yet fully understood,available evidence has highlighted the implication of clathrin-mediated endocytosis as part of thisprocess [123,124].

Regardless of the mechanisms involved in lectin interactions with insect cells, in order to be toxic,a lectin must avoid degradation by the digestive enzymes from the insect gut. The insecticidal effect

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of some lectins is attributed to the resistance of these proteins to proteolysis and to their properties ofstability in a wide pH range. For example, PF2, an insecticidal lectin to Zabrotes subfasciatus, is resistantto in vitro proteolysis by insect digestive enzymes. This is in contrast to PHA-E lectin that is digestedafter 4 h of incubation with these enzymes, and therefore is not toxic for this insect [8]. Other lectins withinsecticidal activity that share the common feature of resistance to proteolysis by different digestiveenzymes include, G. simplicifolia II lectin (GS II), Ulex europeus agglutinin (UEA) and B. monandralectin [117,118]. The degree of resistance to digestive enzymes depends on the capacity of the lectin tobind to glycoconjugates of the insect gut. GS II mutant lectins lacking the ability to bind carbohydratesdisplayed sensitivity to proteolysis by digestive enzymes with a consequent loss of toxicity [125].

Table 2. Receptors of plant lectins of different insects.

Lectin Receptor Insect Reference

Allium sativum L. bulbs(ASAI and ASAII)

Aminopeptidase N Sucrase Acyrthosiphon pisum [126]

Galantus nivalis lectin Ferritin Nilaparvata lungesSpodoptera littoralis

[127][128]

Myracrodruon urundeuvaleaf lectin (MuLL)

Trypsin α-amylase Aedes aegypti [129]

Bauhinia monandra leaflectin (BmoLL)

α-amylase Callosobruchusmaculatus

[117]

Concanavalin A β-glucosidasescathepsin L

Rhopalosiphum padi L. [130]

Allium sativum LeafAgglutinin (ASAL)

(Nicotinamide adeninedinucleotide reduced) quinone

oxidoreductase

Brown planthopper [131]

Colocasia esculenta tuberagglutinin (CEA)

Vacuolar ATP synthaseATP synthase

Heat shock protein 70 clathrinheavy chain

Lipaphis erysimi [122]

Sarcoplasmic endoplasmicreticulum typ Ca2+ATPase

Bemisia tabaci [122]

PF2 lectin α-amylaseV-type proton ATPase

Arginine kinaseProhibitin

PolyubiquitinActin

ATP-dependent RNA helicaseATP synthase subunit alpha

Mitochondrial-processingpeptidaseα-tubulin

Odorant receptorCytochrome c oxidase

Zabrotes subfasciatus [132]

[133]

Allium sativum lectin AminopeptidaseCadherin-N

PolycalinAlkaline phosphatase

Cytochrome P450

HelicoverpaArmigera

[134]

AlanylAminopeptidase N

Sucrase

AcyrthosiphonPisum

[134]

In summary, the insecticidal effect exhibited by several plant lectins is a complex phenomenonthat relies on the capacity of the lectin to interfere with critical physiological processes during insect

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development. Such interference often may be mediated by a lack of nutrient absorption that occurswhen midgut epithelial cells are atrophied as a result of the interaction of lectins with glyco-targetslocated either on the surface or inside the cell. Studies of the recognition of plant lectins by insectgut glyco-targets can allow one to develop hypotheses on which cellular pathways are affected bythe insecticidal activity. These pathways vary depending on the type of insect and the lectin, and couldbe as diverse as the type of recognized glycan or receptors, and might include the effectors of energymetabolism, and redox and ionic homeostasis (summarized in Table 2).

Finally, the study of potential non-yet-identified insecticidal plant lectins may contribute tothe development of distinctive tools for sustainable pest control. However, in this regard, care mustbe taken considering the effect lectins might exert on beneficial insects as well.

6. Antitumor Activity

Recently, an increased interest in the potential of plant lectins as cancer therapeutic agents hasbecome evident. Lectins can be used to study the metastatic distribution patterns, the expressionprofiles of cancer cells glycoconjugates and their biological effects in various tumors. It is well knownthat carbohydrates present on the cell membrane are important for cell recognition, communicationand adhesion. In cancer, these interactions are essential for tumor progression and metastasis. In tumorcells, glycosylation is generally altered in comparison with normal cells and these alterations canbe detected by lectins [135,136]. Litynska et al. compared the lectin-binding pattern in different humanmelanoma cell lines using the PHA-L lectin and other non-legume lectins, including the Galanthusnivalis lectin, S. nigra lectin, MAL, Datura stramonium agglutinin and wheat germ agglutinin (WGA).Studies demonstrated that acquisition of metastatic potential of tumor cells is correlated withthe expression of branched and sialylated complex N-oligosaccharides [137]. Interestingly, the bindingof MAL-I to gastric cancer cells was proportional to their metastatic capacity, and a high expressionof α (2,3)-linked sialic acids was closely correlated with lymph node metastasis [137]. Korourian et al.used G. simplicifolia lectin-I (GS I) and Vicia vilosa agglutinin (VVA) to establish an expressionanalysis of carbohydrate antigens in human breast ductal carcinoma in situ (DCIS). Both lectinsshowed a significant association with nuclear grade (cell size and uniformity) of DCIS [138]. Positivecorrelations also were found between the expression of VVA- and GSI-reactive structures and tumorgrade in DCIS patients, suggesting that the glycoconjugates found in these antigens are associated withinvasiveness in DCIS [138]. PHA can be used to discriminate between hepatocellular carcinoma andbenign liver disease [139]. This suggests that lectins could potentially be used to develop prognostictools for cancer diagnosis and the detection of metastasis, and to some extent to estimate the severityof the clinical case.

In addition to these uses, plant lectins have shown in vivo and in vitro antitumor activity. Lectinswith the potential to induce inhibition of tumor cell growth, such as, Con A, Mistletoe type-I lectins andPHA are currently in different phases of clinical trials [138,140]. Plant lectins can modify the expressionof interleukins and some protein kinases and in this way modulate the immune system. Furthermore,in cancers, lectins could alter the signaling pathways involved in the expression of members ofthe Bcl-2, Autophagy (ATG) related, and caspase families, as well as p53, ERK, Ras-Raf, and BNIP3,and thereby induce both apoptosis and autophagy [141]. Soybean lectin produces reactive oxygenspecies in a dose-dependent manner in HeLa cells inducing apoptosis, autophagy and DNA damagein the cells [142].

The cell surface carbohydrates are key targets for lectins; in cancer, a general pathway involvesrecognition of carbohydrate receptors triggering the activation of enzymes such as MBL-associatedserine proteases [143]. However, the binding of lectins to glycans on the cell membrane may notbe sufficient to induce apoptosis. Kim et al. showed that cell death requires lectin endocytosis thattriggers signaling for apoptosis [143]. Con A is cytotoxic to hepatoma cell lines. Its toxicity is mediatedprimarily by its binding to the mannose moiety of cell membrane glycoproteins, with subsequentinternalization and accumulation in the mitochondria. Autophagy is then activated, leading to

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lysosomal degradation of the affected mitochondria and, ultimately, cell death [144,145]. Overall,these findings of cancer research have revealed the mechanisms responsible for the antitumor actionof different plant lectins. These mechanisms are varied and depend upon different factors such asthe tumor cell origin and type as well as lectin concentration.

7. Conclusions

The screening and characterization of novel lectins from legume resources has allowedthe discovery of proteins with a great capacity to distinguish specific glycans from a diversityof glyco-targets found in a variety of organisms. Legume lectins could be potential candidatesfor developing tools based on protein–carbohydrate interactions with variable specificities.Lectin-mediated drugs focused on targeting specific cells could lead to promising anticancer andantimicrobial treatments, which would directly impact areas of economic importance, such asthe pharmaceutical and food industries and agriculture. Additionally, the use of lectins againstinsect pests could provide a form of sustainable pest control.

More research is needed to explore and support the therapeutic effect of lectins. Studies of actionmechanisms, the relationship between the role of the lectins and their molecular features, and finally,their effect in modulating the expression of proteins and genes are required to move forward the useof lectins for clinical applications.

Domesticated legumes provide an accessible and abundant source of lectins, unlike wild legumes,which, in some regions, may even be considered protected species. In some cases, successfulrecombinant production of lectins would be a key factor in whether a specific lectin could finduse in a feasible industrial application. The overexpression of recombinant lectins remains a greatchallenge, even more so for those lectins where the lack of adequate post-translational modificationscould compromise their activity.

Acknowledgments: The authors thank Joy Winzerling for her critical reading and editing of the manuscript.

Author Contributions: All authors compiled the information, wrote the review, read and approved thefinal manuscript.

Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

CRD Carbohydrate recognition domainPM Peritrophic membranePG Peritrophic gelPHA Phaseolous vulgaris agglutininMAL-1 Maackia amurensis lectin 1DCIS Human breast ductal carcinoma in situGS IV Griffonia simplicifolia IV lectinMAH and MAL-1 Maackia amurensis lectinsVLs Acacia constricta isolectinsWGA Wheat germ agglutininLCA Lens culinaris agglutininPSA Pisum sativum agglutininGS II Griffonia simplicifolia II lectinUEA Ulex europeus agglutininASAI and ASAII Allium sativum L. bulbsMuLL Myracrodruon urundeuva leaf lectinBmoLL Bauhinia monandra leaf lectinASAL Allium sativum Leaf AgglutininCEA Colocasia esculenta tuber agglutininGS I Griffonia simplicifolia lectin-IVVA Vicia vilosa agglutinin

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